This is a cached page for the URL (http://www.upstate.edu/biochem/amberg/protocols/smash_and_grab.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Smash and Grab

 

PROTOCOLS

 

Smash and Grab

David Amberg

  1. Grow cells in 3mls selective media o/n.
  2. Pellet cells by 2 quick spins in a microfuge.
  3. Resuspend cells in 200ul of the following soln:
    • 5ml SCE
    • 60ul 10mg/ml 100T Zymolyase
    • 10ul 2-ME
  4. Vortex and inc at 37¡C for 30-60 min.
  5. Add 400ul 0.2N NaOH/1%SDS, invert to mix and inc. on ice for 5 min.
  6. Add 300ul 3M K/5M OAc pH4.5, invert to mix and inc on ice x 5 min.
  7. Spin 2 min in microfuge, pour super into a fresh tube and repeat spin. Remove 500ul to a fresh tube.
  8. Add 300ul iPrOH, vortex and let stand for 5 min. at RT.
  9. Pellet DNA 5 min in a microfuge, then wash pellet with 70% EtOH and repeat spin.
  10. Dry pellet in speed vac and recon in 25ul TE.
  11. Porate 2ul into electrocompetent coli, recover and plate all.


| Amberg Lab Home Page | Research | People | Publications | Protocols | Other Links | Upstate Medical University |

| UMU College Of Gradutate Studies | The Colleges at UMU | University Hospital | Research Development | UMU Library |




Site designed by: David C. Amberg
Site maintained by: Blaine Bettinger
Department of Biochemistry and Molecular Biology
Last updated: November 2, 2001
All contents copyright© 2000
SUNY Upstate Medical University

750 East Adams Street
Syracuse, NY 13210